Arrestin was cloned into the pCITE-4a vector (Novagen) to allow for transcription from a T7 promoter. The TNT Quick Coupled Transcription and Translation kit was used according to the manufacturer’s protocol (Promega), to express 35S-methionine-labelled WT arrestin and 3A_arrestin (L374A, V376A, and F376A). Radiolabelled WT arrestin and mutant arrestin proteins were incubated with His8-MBP-rhodopsin fusion protein immobilized to 50 μl of maltose agarose bead suspension. Proteins and beads were incubated at 4 °C for one hour on a rotating platform in binding buffer containing 20 mM Tris-HCl, pH 7.4, 100 mM NaCl and 0.02% DDM/0.004% CHS. The beads were then washed three times with binding buffer and resuspended in 400 μl elution buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.02% DDM/0.004% CHS and 100 mM maltose). The eluates were concentrated and incubated at room temperature for 15 minutes with 2 × loading dye. Samples were separated on 12% sodium dodecyl sulfate (SDS)-denaturing polyacrylamide gels. Gels were stained with Coomassie R-250, dried at 70 °C for 90 minutes, and exposed overnight to a phosphor storage screen. Results were visualized on a PhosphorImager (Fuji).
Tnt quick coupled transcription and translation kit
Manufactured by Promega
The TNT Quick Coupled Transcription and Translation kit is a laboratory tool that enables the rapid in vitro synthesis of proteins from DNA templates. The kit provides the necessary components for the coupled transcription and translation processes, allowing for the efficient production of recombinant proteins.
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4 protocols using tnt quick coupled transcription and translation kit
1
Arrestin Binding to Rhodopsin Assay
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Arrestin Binding to Rhodopsin Assay
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Cell-free Protein Import into Mitochondria
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Proteins were synthesized from pGEM4 plasmid templates and radiolabeled with 35S methionine in a cell-free translation system (TNT Quick Coupled Transcription and Translation kit, Promega).
Import reactions were essentially performed as described previously (Peleh et al., 2015 (link)) in the following import buffer: 500 mM sorbitol, 50 mM HEPES, 80 mM KCl, 10 mM magnesium acetate, and 2 mM KH2PO4, pH 7.4. Mitochondria were energized by addition of 2 mM ATP and 2 mM NADH before radiolabeled precursor proteins were added. To dissipate the membrane potential, a mixture of 1 μg/ml valinomycin, 8.8 μg/ml antimycin, and 17 μg/ml oligomycin was added to the mitochondria. Precursor proteins were incubated with mitochondria for different times at 25°C before nonimported protein was degraded by addition of 100 μg/ml proteinase K. For import assays into mas1ts mitochondria, mitochondria were incubated in import buffer for 20 min at 37°C to inactivate MPP before radiolabeled precursor proteins were added to the reaction. The import was also performed at 37°C.
Import reactions were essentially performed as described previously (Peleh et al., 2015 (link)) in the following import buffer: 500 mM sorbitol, 50 mM HEPES, 80 mM KCl, 10 mM magnesium acetate, and 2 mM KH2PO4, pH 7.4. Mitochondria were energized by addition of 2 mM ATP and 2 mM NADH before radiolabeled precursor proteins were added. To dissipate the membrane potential, a mixture of 1 μg/ml valinomycin, 8.8 μg/ml antimycin, and 17 μg/ml oligomycin was added to the mitochondria. Precursor proteins were incubated with mitochondria for different times at 25°C before nonimported protein was degraded by addition of 100 μg/ml proteinase K. For import assays into mas1ts mitochondria, mitochondria were incubated in import buffer for 20 min at 37°C to inactivate MPP before radiolabeled precursor proteins were added to the reaction. The import was also performed at 37°C.
4
In Vitro Protein Synthesis from Yeast DNA
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For in vitro protein synthesis, plasmid-based templates or PCR templates, generated by using genomic yeast DNA as template, were used that contain a SP6 promoter and a ribosomal binding site in front of the open reading frame. In case of FIS1 and TOM22, additional codons for methionine residues were added before the STOP codon to increase the signal intensity. For PCR templates, transcripts were generated using the mMESSAGE mMACHINE SP6 kit (Thermo Fisher) and purified using the MEGAclear Transcription clean-up kit (Thermo Fisher). Proteins were radiolabeled with [ 35 S]methionine in a cell free translation system based on reticulocyte lysate (TNT Quick Coupled Transcription and Translation kit and Flexi Rabbit reticulocyte lysate (Promega)) following the manufacturer's recommendations.
Cell Reports 31, 107567, April 28, 2020 e3
Cell Reports 31, 107567, April 28, 2020 e3
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